Method of purifying streptomycin



Patented Sept. 2, 1952 Walter A. Winston, Forest Hills, N. Y., assignor I"..to"SchenleyIndustries, Inc acorporation of r lawar No Application October 2, .SerialNo. 52,590

E 9 Claims. (01. 260-210)- My invention relates to improved streptomycin preparation. s

The production, concentration,--and purification of; streptomycin ,are well known, and disclosed, for example, at pages 1335-4336 of the 24th edition"(1947) of The Dispensatory of the United States of America.

.As' stated, in this publication, streptomycin must be purified sothat it contains nohistamine or histamine-like substances, which act to depress;

blood :pressure. Such histamine-like substances are usually; produced during the production of streptomycinby Streptomyces griseus.

The standard test for such elimination of histamine and'histamine-like substances,is to narcotize-a subject cat withusodium, pentobarbital;

and to inject the streptomycin or a. complex thereofginto'the cat; at-a level of 3000 units per kilogram of the body weight of the subject. Such unit is the antibiotic activityof onemicrogram of the base of streptomycin. 'A streptomycin preparation is considered-suitable; for injection into .humansubjects if its content of histaminelike substances has vbeen so. reduced that the injection at, said level produces a lowering of blood pressure in the subject cat; which is equal to or less than the lowering of blood pressure which is produced-by Q,1;microgram of histamine per kilogram, of the body Weight of the subject cat.

1 I have discovered thatl canreduce the concentration ofliistamine or histamine-like substances in streptomycin preparations, to and below said test level, by reacting a solution of impure streptomycin, preferably an aqueous ;solution,- with.

bromine, preferably at a pHbelow 7. The reaction maybe conducted at ordinary room temperature of 20 C.-25 C., and at standard atmospheric pressure of 760, mm. of mercury. The 1 recovery of streptomycin from the reaction is e histamine or histamine-like substances, with the resultant formation of hydrobromic acid. However, the reaction may resultjin,bromination.of the histamine or histamine-likeimpurity;

As a starting material, I prefer to usean aque- I one solution of impure streptomycin whichhas a pH below '7;, as 5.7. ;This is not necessary when bromine is used, because the reaction with broa new and improved. method ofpurifyingstreptomycin and a new and V do not however'by themselves serve "to free st're mine quickly lowers the initial pH, which may be 7, to a value below 7. I prefer to maintain a pH below 7 in the reaction. solution'du'ring -the entire period of the reaction, or commencing very shortly after the beginning of the reaction period.

Further objects and featuresand alternatives of my invention are disclosed in the followingdescription, which discloses-illustrative examples to which the invention is not limited.

' Starting material No. 1

When streptomycin is produced by a fermenta 1 tion of a suitable medium with S. 'gTz'seua 'the histamine-like substance is also prese'nt in'the' iinal resultant crude fermentation broth. In obtaining streptomycin from such a broth, sub' stantially free of histamine like impurities, I find it convenient to prepare a concentrate of streptomycin by a sequence orwellknown steps which tomycin from histamine-like impuri-ties." After' producing such a streptomycin concentrate l" thentreatit according to my invention'in order to remove and/or destroy the histamine-like impurity Without affecting or destroying to any substantial degree the streptomycinitself;

There are various well known methods of con-*- centrating the original impure 'or crude culture broth. H i I As one example, the streptomycin can'be'" adsorbed on silica gel irom the original crude broth. Such gel is separated andwashed with w'aterwhich hasfbeen acidified withihydrochloric I acid or other suitable acid; thus eluting the adsorbed streptomycin as a hydrochloridesalu'lii hydrochloric acid is used. The resultant elution liquid is neutralized. The streptomycin is" adsorbed onfactivated carbon from the neutral elution liquid, and this activated carbonis' separated: by centrifugation or filtration. 'Ih'e streptomycin" is eluted from the activated carbonwith fagueous V acetone which is acidified'withl' ydrochloric' acid or other suitable acid; lhe elution' liquid isQneutralized and then concentrated any onvene tional method, to givea preparation containing 68,000 units per ml. of streptomycin.

The resultant neutral concentrate may be used as one of the reactants, or: said neutral concem trate maybeacidifiedprior to the-reaction,.as to. a pH of 5.7. If it is desired to acidifysa id neutrali concentrate prior to the reaction, it may be acidi fied with hydrochloric acid. 3

Starting material No? 2 As an alternativalcan adsorb the streptomycin w from the original crude fermentation broth by j. H a 2,609,369

Bromine reactant No. 1

This is bromine water, of the kind which is used as a bromine test solution. It contains 2 cc. of bromine, dissolved in 100 cc. of :distilled water.

Bromine reactant .No. 2

'onactivated: carbon. This activated carbon is This is a solution of bromine in chloroformor other organic solvent.

Example No. 1-

The reactants are starting material No. 1, and

said bromine water, in the proportion of :5 cc. of starting material No. 1 (68,000 units per ml. of streptomycin) Y'tocc. of ,said brominewater. As'above'noted,:saidstarting material-No. 1 may be neutral or, it may have a pH -below 7, such asa pI:I-0f..5.7, whenit is used as a reactant.

These reactants are intermixedand reacted ford 15.:minutesat normal room temperatureof C.- -C.,. at said normal atmospheric pressure. The mixture-is optionally continuously mechanicallyagitated during this reaction period of-15 minutes.

During the reaction, if the initial pH of starting material No. 1 is 5.7, the pH of the reaction mass ormixtureidrops to- 2.1-.

. The reaction mass is then neutralized-by means of sodium hydroxide.

When-thus neutralized, a transient purple color is-oftenpbservedr. This color soon becomes a dull color. 1:

Excess :freebromine. is thenremoved from the neutral aqueous solution of thepurified strepto-.

mycin,. by extraction with chloroform, in the proportion:.of20 cc. of chloroform per 15 cc. of bromineiwater. This is done at normal room temperature andaat normalpressure. The solution ofthebromine in the chloroform is separated from the-aqueous streptomycin solution byusual the purified streptomycin are removed as in Example No. l, and the setting step of Example No. 1 is used, with removal of resultant sediment.

Example N o. 3

Starting material No. 2 is used. Otherwise, the procedure the same as -in-i-ExamplesiNo. 1 or No. 2.

The streptomycin is then adsorbed from the neutral aqueous solution of purified streptomycin separated and eluted with acidified aqueous acetoneandthe elution liquid may be neutralized as the end-product, which may be concentrated or otherwise treated." This produces a purer endproduct. Also, various impurities, such as inorganic bromides and brominated impurities, are

2 not adsorbed on the activated carbon.

The aqueous :solution of streptomycinis then allowedto-stand at 5 C-. 'during,a settling period 0f..24 hours.

purified. streptomycin.

Streptomycinrwhich has been thus. treated...

passes the above-mentioned .test for histaminelike substances, and it .may be used, for injection purposesiormaking complexes, etc.

'Tests have shown that from193 %-100% by weight 'of the streptomycin inthe originalimpure starting materialremains. undecomposed inthe.

aqueous solutionof the purified streptomycimby. following this example.

:Example N0. 2

Sedimentsettles out and this: is. separatedr ,from,..,the. neutral aqueous solution of.

' It"is thus advantageous to use the activated carbon as an adsorption agent, after the reaction with the bromine.

As above notedQthe starting material may be an aqueous solution of streptomycinof anycon' centration and purity.

Bromine has an extremely advantageous and"-' selective action forthe-purposes of my invention.-

Iodine does=- not react with the histamine like i;

substances.

Sodium -hypochlorite,-NaOCl, is no more efiective than bromine, and: it results ina greater loss of streptomycin.-

Whensodium hypochlorite is 'used, the start in'g materialis preferably acidified, as. with=hypo" chloric acid, thus forming 'hypochlo'rous acid; in;

' the reaction mixture: The oxidizing action of sodium 'hypochl'orite in aqueous solution, especiallyinacidified aqueous solution,-is well known:

I have described-preferred embodiments of my invention; but numerous changes and omissions! and additions can be made without 'departing from its scope;

I claim:

1. Process for reducing the histaminic activity'i of histamine and histamine-likeimpurities in an aqueous streptomycin-containing solution thatcomprises treating said 'so'lution with an' excess of a solution' of bromine under non-alka' line 'conditions at a temperature approximating ordinary 1 roomtemperature'' and atmospheric pressure, for a period of about 15 1 minutes,-- ad justing tlie #hydro'gen ion concentration ofthe mixtureto a pH- approximating-7 and removing the excess bromine present I 2. Process as defin'ed in -claim '1 wherein the so'lution of bromine used is an-aqueous solution of elemental bromine. p

3; Process as defined in claim 1 wherein the solution ofbromine =usedis an aqueous solution of elemental bromineycontaining about '2 cc-Jof f elemental bromine per cc; of water.

4. Process as defined in claim 1 wherein the; solution ofbromine is one in whichthe-solvent is an organic liquid.

5f Process as defined in claim 1 'wl'ierein -the solution of bromine is one in which the-solyent is chloroform.

6. Process as defined in claim l wherein ith excess of bromine present is 'removedby extraction with a water-immiscible "organic solvent capabl'eof dissolving'bromine.--

'7. Process as d'efined inpIaim -I wh'erei-n' the? excess of" bromine present: is :removed by *exifrafc tion With'chIOrGfOrmJ 1 8:5 Process as defined-in claim 1 further "characterized in that the streptomycin content ofthefinal neutralized solution is recovered from the solution by adsorption on activated charcoal, followed by elution with an acidic organic solvent.

9. Process as defined in claim 1 further characterized in that the streptomycin content of the final neutralized solution is recovered from the solution by adsorption on activated charcoal, followed by elution with an acidic aqueous acetone mixture.

WALTER A. WINSTEN.

6 REFERENCES CITED The following references are of record in the file of this patent:

Fried et al., JACS, v. 69 (1947), pp. 79-86 8 pages.

Van Dolah et al., Arch. Biochem, v. 12 (1947), pp. 7-12, 6 pages. 

1. PROCESS FOR REDUCING THE HISTAMINIC ACTIVITY OF HISTAMINE AND HISTAMINE-LIKE IMPURITIES IN AN AQUEOUS STREPTOMYCIN-CONTAINING SOLUTION THAT COMPRISES TREATING SAID SOLUTION WITH AN EXCESS OF A SOLUTION OF BROMINE UNDER NON-ALKALINE CONDITIONS AT A TEMPERATURE APPROXIMATING ORDINARY ROOM TEMPERATURE AND ATMOSPHERIC PRESSURE, FOR A PERIOD OF ABOUT 15 MINUTES, ADJUSTING THE HYDROGEN ION CONCENTRATION OF THE MIXTURE OF A PH APPROXIMATING 7, AND REMOVING THE EXCESS BROMINE PRESENT. 